Phosphoinositides (PIs) are lipid second messengers implicated in membrane remodeling. PtdIns3P and PtdIns(3,5)P2 are present on endosomal compartments where they play an important role in membrane trafficking. In addition, an increasing number of human genetic diseases (including myopathy and neuropathies) are associated to mutations in enzymes regulating the turnover of these endosomal PIs. PtdIns5P is far less characterized but it has been implicated in signal transduction and vesicular transport. Recently, two PtdIns(4,5)P2 4-Ptases (PtdIns(4,5)P2 4-Ptase type I and II) that catalyze the hydrolysis of PtdIns(4,5)P2 to PtdIns5P have been identified. We found that over-expression of either PtdIns(4,5)P2 4-Ptase I or II, induced a time-dependent retrograde aggregation of late endosomes/lysosomes over the microtubule-organizing center (MTOC) but had no effect on early endosomes or Golgi. Retrograde trafficking of lysosomes was inhibited by nocodazol or by expression of dynamitin, indicating that microtubules and dynein motors are required for the aggregation. Lysosomal clustering led to delayed degradation of EGFR, defective autophagosome maturation, and secretion of lysosomal hydrolases, thus suggesting a role of PtdIns5P in membrane trafficking. Moreover, simultaneous depletion of both, endogenous PtdIns(4,5)P2 4-Ptase I and II induced dispersion of lysosomes to the cell periphery. One of the problems in studying the function of PIs is that prolonged over-expression of PI-kinases or phosphatases may affect multiple signaling and trafficking events. To overcome this problem we modified the previously described rapamycin/FRB/FKBP system to attain inducible recruitment of the catalytic domain of PtdIns(4,5)P2 4-Ptase I to late endosomes/lysosomes upon addition of rapamycin. This system will allow monitoring the effect of changes in the levels of PtdIns5P in the distribution of endosomes in real time.